Our published experiments have indicated that the DNA demethylating agent 5 aza 2 deoxycytidine (DAC) mediates dramatic induction of CT-X genes such as NY-ESO-1, MAGE-A1, and MAGE-A3 in cultured lung cancer cells, but not normal human bronchial epithelia (NHBE). Furthermore we have demonstrated that the HDAC inhibitor Depsipeptide FK228 (DP) enhances DAC-mediated induction of CT-X genes in lung cancer cells. Induction of CT-X gene expression by sequential DAC/DP treatment coincided with a dramatic increase in BORIS expression in lung cancer cells. Methylation specific PCR (MSP) and sodium bisulfite sequencing experiments demonstrated that activation of BORIS in cultured lung cancer cells coincided with demethylation of a CpG island within the BORIS promoter. Additional MSP experiments revealed de-repression of BORIS in 6 of 12 primary lung cancer specimens; in several patients, de-repression of BORIS was detected in lung cancers as well as histologically normal tissue adjacent to these neoplasms, raising the possibility that activation of BORIS is an early epigenetic event during pulmonary carcinogenesis. Subsequent electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that BORIS physically interacts with the NY-ESO-1 promoter, and that de-repression of NY-ESO-1 in lung cancer cells coincides with a CTCF-to-BORIS shift in occupancy within the NY-ESO-1 promoter. A similar phenomenon was observed for MAGE-A1 activation in lung cancer cells. Additional studies were undertaken to further examine the mechanisms by which BORIS contributes to transcriptional regulation of NY-ESO-1. Software guided analysis of the NY-ESO-1 promoter region revealed a variety of potential transcription factor binding sites including several SP1 motifs. Western blot experiments demonstrated that sequential DAC/DP treatment enhanced nuclear translocation of SP1 in lung cancer cells. Transient transfection assays using deletion mutant promoter-reporter constructs, as well as EMSA and ChIP experiments demonstrated that NY-ESO-1 promoter activity coincided with occupancy of the proximal SP1 binding site in lung cancer cells. Mutations within the SP1 recognition sequence eliminated binding of SP1 to this motif in vitro, and markedly diminished NY-ESO-1 promoter activity in vivo (Figure 1). Additional experiments using siRNA techniques revealed that inhibition of SP1 expression decreased NY-ESO-1 promoter activity, whereas knock-down of CTCF augmented expression of this CT-X gene in lung cancer cells. Immunoprecipitation experiments demonstrated that SP1 physically interacts with BORIS, but not with CTCF in vivo. Collectively these data suggested that BORIS recruits SP1 to de-repress NY-ESO-1 (and possibly other CT-X genes) in lung cancer cells. Because histologically normal epithelia as well as premalignant lesions adjacent to established lung cancers exhibit de-repression of BORIS, it is conceivable that de-repression of this germ cell transcription factor contributes to initiation and maintenance of the transformed phenotype of lung cancer cells. As such, a series of experiments were initiated to comprehensively investigate the effects of BORIS expression in SV40- or cdk4/h-tert- immortalized human bronchial epithelial cells (BEAS, and HBEC, respectively), which are deficient for BORIS and CT-X gene expression, as well as Calu-6 (p53 null) and A549 (p53 wt) lung cancer cells exhibiting extremely low level BORIS and CT-X gene expression. Interestingly, BEAS cells transduced with BORIS exhibited loss of contact inhibition, increased proliferation and invasion, as well as enhanced clonogenic potential following tetracycline exposure relative to control cells. This phenomenon appeared to coincide with silencing of E-cadherin, a gene known to regulate epithelial-mesenchymal transition, which has been implicated in cancer metastasis. Additional studies are in progress to further evaluate the effects of BORIS expression regarding proliferation, tumorigenicity, and chemo/XRT sensitivity of lung cancer cells. These studied could reveal novel epigenetic biomarkers of prognosis in lung cancer patients.